Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF‐β1 and TNF‐α. Protein levels of α‐smooth muscle actin (α‐SMA), type I collagen, heat shock protein‐47 (HSP‐47), fibronectin (FN), ED‐A‐FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α‐SMA and actin fibers were identified by immunofluorescence. TGF‐β1, TNF‐α, and α‐SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF‐β1 activity was evaluated using a plasminogen activator inhibitor‐1 (PAI‐1) reporter transfected in HGFs. Results: TGF‐β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α‐SMA and ED‐A‐FN. Moreover, TGF‐β1 induced the production of type I collagen, HSP‐47, FN, and periostin. Costimulation with TNF‐α and TGF‐β1 significantly reduced the expression of all the above‐mentioned proteins. TNF‐α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI‐1 reporter. Conclusions: TNF‐α inhibits several cell responses induced by TGF‐β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.     

Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor

Background: Transforming growth factor‐β1 (TGF‐β1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF‐β1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF‐β1–mediated myofibroblast transformation. Methods: To study the inhibitory effect of Smad7 on TGF‐β1/CTGF‐mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α‐SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme‐linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGF's role on myofibroblast transformation was examined by enzyme‐linked immunosorbent assay and small interference RNA. Results: TGF‐β1 induced the expression of α‐SMA and CTGF, and small interference RNA–mediating CTGF silencing prevented fibroblast‐myofibroblast switch induced by TGF‐β1. In Smad7‐overexpressing fibroblasts, ablation of TGF‐β1–induced Smad2 phosphorylation marked decreased α‐SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α‐SMA and phospho‐Smad2 and significant reduction on CTGF and type I collagen production. Conclusions: CTGF is critical for TGF‐β1–induced gingival fibroblast‐myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.     

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts

Agis H, Watzek G, Gruber R. Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor by periodontal fibroblasts. J Periodont Res 2012; 47: 165–173. © 2011 John Wiley & Sons A/S Background and Objective: Pharmacological inhibitors of prolyl hydroxylases (PHDs) can induce a proangiogenic response that favors wound healing and bone regeneration. However, the response of periodontal cells to PHD inhibitors is unknown. Material and Methods: To determine the effects of PHD inhibitors on periodontal cells, we exposed human fibroblasts from the gingiva and the periodontal ligament to dimethyloxallyl glycine, desferrioxamine, l ‐mimosine and CoCl₂. Viability, proliferation, and protein synthesis were assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), [³H]thymidine, and [³H]leucine incorporation, respectively. The levels of Ki67, hypoxia‐inducible factor 1α (HIF‐1α), p27, phosphorylated c‐Jun N‐terminal kinase (JNK) and phosphorylated p38 were determined by immunohistochemistry and western blotting. Vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative PCR. Protein levels of VEGF and interleukin (IL)‐6 were evaluated by immunoassays. Results: We found that PHD inhibitors, while leaving cell viability unchanged, reduced proliferation and protein synthesis. This was paralleled by decreased Ki67 levels and increased p27 levels, suggesting that PHD inhibitors provoke growth arrest. Independently from this response, PHD inhibitors stabilized HIF‐1α and increased the production of VEGF. This increase of VEGF was observed in the presence of proinflammatory IL‐1 and pharmacological inhibitors of JNK and p38 signaling. Moreover, PHD inhibitors did not modulate expression of IL‐6 and the phosphorylation of JNK and p38. Conclusion: These results suggest that PHD inhibitors enhance the production of VEGF in periodontal fibroblasts, even in the presence of proinflammatory IL‐1. The data further suggest that PHD inhibitors do not provoke a significant proinflammatory or anti‐inflammatory response in this in vitro setting.     

Collagen Membranes Adsorb the Transforming Growth Factor‐β Receptor I Kinase‐Dependent Activity of Enamel Matrix Derivative

Background: Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)‐β signaling in oral fibroblasts. Methods: Three commercially available collagen products were exposed to EMD or recombinant TGF‐β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF‐β target genes interleukin (IL)‐11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. Results: EMD or TGF‐β1 provoked a significant increase of IL‐11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF‐β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin‐coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL‐11 and PRG4. Conclusion: These in vitro findings suggest that collagen products adsorb a TGF‐β receptor I kinase‐dependent activity of EMD and make it available for potential target cells.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

The expressions of IGF‐1, BMP‐2 and TGF‐β1 in cartilage of condylar hyperplasia

Summary Condylar hyperplasia is a complex post‐natal growth abnormality of the mandible and condyle, which leads to facial asymmetry. We investigated the distributions of insulin‐like growth factors (IGF‐1), bone morphogenetic protein‐2 (BMP‐2) and transforming growth factor‐β1 (TGF‐β1) in cartilage of condylar hyperplasia and revealed relationships between age and the cartilaginous thickness. Twenty patients with condylar hyperplasia were divided into four histopathological types. The cartilaginous thickness and age in different histological types were analysed, and the localizations of IGF‐1, BMP‐2 and TGF‐β1 were detected by immunohistochemistry analysis. The cartilaginous thickness of condylar hyperplasia significantly increased. The cartilaginous thickness of type III was significantly thicker than type I and type II, Bivariate correlation revealed a significant correlations between age and the cartilaginous thickness (r = 0·68, P = 0·01). However, the expressions of IGF‐1, BMP‐2 and TGF‐β1 were the strongest in type I. In almost all types of condylar hyperplasia, the presence of IGF‐1 and BMP‐2 was found mainly in the proliferative chondrocyte layer and the hypertrophic chondrocyte layer, and only a few in the calcified chondrocyte layer. The presence of TGF‐β1 widely distributed from the fibrous articular surface to the calcified cartilage. These findings suggest that the proliferative activity of cartilage in condylar hyperplasia is strongly associated with age and cartilaginous thickness. Therefore, the four pathological types of condylar hyperplasia seem more likely to be four discontinuous stages.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Molecular events associated with ciclosporin A-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts

Sobral LM, Aseredo F, Agostini M, Bufalino A, Pereira MCC, Graner E, Coletta RD. Molecular events associated with ciclosporin A‐induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts. J Periodont Res 2012; 47: 149–158. © 2011 John Wiley & Sons A/S Background and Objective: Ciclosporin A (CsA)‐induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor‐β1 (TGF‐β1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF‐β1 signaling inhibitor, in the events associated with CsA‐induced extracellular matrix accumulation was performed. Material and Methods: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7‐overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP‐2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho‐Smad2 were performed to measure the activation of TGF‐β1 signaling. Results: Although the treatment with CsA stimulated TGF‐β1 production in both control and Smad7‐overexpressing fibroblasts, its signaling was markedly inhibited in Smad7‐overexpressing cells, as revealed by low levels of phospho‐Smad2. In Smad7‐overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP‐2 were significantly blocked. Smad7 overexpression blocked CsA‐induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. Conclusion: The data presented here confirm that TGF‐β1 expression is related to the molecular events associated with CsA‐induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP‐2 activity.     

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.     

TGF‐β1 regulates the invasive and metastatic potential of mucoepidermoid carcinoma cells

J Oral Pathol Med (2011) 40 : 762–768 Background: Patients with mucoepidermoid carcinoma exhibit poor long‐term prognosis because of the lack of therapeutic strategies that effectively block tumor progression. We have previously characterized the Ms cells as a highly metastatic mucoepidermoid carcinoma cell line that expresses high levels of transforming growth factor β1 (TGF‐β1). Here, we studied the effect of suppressing TGF‐β1 by RNA silencing on the invasive and metastatic potential of mucoepidermoid carcinoma. Methods: Cell motility, substratum adhesion, and transmembrane invasion were estimated by migration, matrigel adhesion, and matrigel invasion assay. Matrix metalloproteinase (MMP)‐2 and MMP‐9 activity were determined using gelatin gel zymography. Balb/c nu/nu nude mice lung metastatic model was used to test the metastatic ability of the Ms cells. Lung metastatic tumors were experimentally induced by mice tail vein inoculation of cancer cells. Results: TGF‐β1 silencing inhibits cell motility, substratum adhesion, and transmembrane invasion. In vivo, a significant decrease in lung metastasis was observed when mice received tail vein injections of TGF‐β1‐silenced mucoepidermoid carcinoma cells, as compared to controls. Conclusion: These results unveil a critical role for TGF‐β1 in the progression of mucoepidermoid carcinomas and suggest that patients with this malignancy may benefit from therapeutic inhibition of the effectors of the TGF‐β1 pathway.     

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor‐β supergene family

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor‐β (TGF‐β) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF‐β₃ (hTGF‐β₃) in growth factor‐reduced Matrigel^® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non‐human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF‐β₃ in Matrigel^® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF‐β₃ in Matrigel^® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF‐β supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine‐tuning of speciation‐related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.     

β2‐Glycoprotein I‐Dependent Anti‐Cardiolipin Antibodies Associated With Periodontitis in Patients With Systemic Lupus Erythematosus

Background : It was reported that patients with systemic lupus erythematosus (SLE) exhibited increased levels of anticardiolipin (anti‐CL) antibodies, a class of antiphospholipid antibodies associated with thrombosis. β2‐glycoprotein I (β2GPI) has been considered as the actual target antigen for anti‐CL antibodies. This study investigates the association of periodontal infection with anti‐CL antibodies in patients with SLE. Methods: Fifty‐three SLE female patients and 56 healthy female volunteers were recruited in this case‐control study. All participants received periodontal examinations. The presence of Porphyromonas gingivalis and Treponema denticola in saliva and plaque samples was detected by polymerase chain reaction. Levels of serum anti‐CL and anti‐β2GPI antibodies were examined using enzyme‐linked immunosorbent assay. Results: Patients with SLE exhibited more periodontal attachment loss and increased titers of serum anti‐CL and anti‐β2GPI antibodies compared with healthy controls. Patients with active SLE who harbored P. gingivalis or P. gingivalis together with T. denticola intraorally exhibited significantly higher anti‐CL and anti‐β2GPI antibodies than those without these bacteria. Anti‐CL and anti‐β2GPI antibody levels correlated positively with clinical attachment level. Furthermore, increased anti‐β2GPI antibody levels were significantly associated with C‐reactive protein and erythrocyte sedimentation rate. Conclusions: Elevated anti‐CL and anti‐β2GPI antibody levels were associated with periodontopathic bacteria and periodontal breakdown in patients with SLE. Periodontitis might be a modifiable risk factor for SLE.     

Orthodontic Force Increases Interleukin‐1β and Tumor Necrosis Factor‐α Expression and Alveolar Bone Loss in Periodontitis

Background: The present study aims to evaluate the effects of orthodontic movement (OM) on the periodontal tissues of rats with ligature‐induced periodontal disease. Methods: Eighty‐eight rats were divided into four groups: 1) negative control (sham operated); 2) periodontal disease; 3) OM; and 4) periodontal disease followed by OM (OMP). Rats were sacrificed 3 hours or 1, 3, or 7 days after OM commencement. Bone volume fraction (BVF) and bone mineral density (BMD) were assessed in hemimaxillae by microcomputed tomography analysis. Expression of the proinflammatory cytokines interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α were evaluated in gingival samples by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, and in the furcation region by immunohistochemistry analysis (IHC). Results: The OMP group had lower BVF and BMD levels compared to the other groups at day 7 (P <0.05). Maximum messenger ribonucleic acid expression of both cytokines was observed in the OMP group at day 1 (P <0.05). In the same period, all proteins were expressed in high levels for all test groups compared to the control group. The number of cells positive for IL‐1β and TNF‐α by IHC was highest in the OMP group at day 1, with progressive reduction thereafter. Conclusion: The results suggest that OM acts synergistically with periodontal disease in periodontal breakdown through upregulation of proinflammatory cytokines.